Yonghaparkia Aerolata sp. nov., a Novel Member of the Genus Yonghaparkia, Isolated From Electronic Waste Associated Bioaerosols

A Gram-positive, non-motile, non-spore-forming and short rod-shaped actinomycete strain, designated GA224 T , was isolated from an electronic waste associated bioaerosols. The isolate is facultatively anaerobic, which is able to grow at 25–40 ℃ (optimum 37 ℃ ) and pH 6.5–8.5 (optimum 8.0). The diamino acid in the cell wall of strain GA224 T is 2,4-diaminobutyric acid (DAB), while major menaquinone is MK-12. The polar lipid prole is composed of diphosphatidylglycerol, phosphatidylglycerol, unidentied phospholipids, unidentied glycolipids and unidentied lipid. The major cellular fatty acid is anteiso-C 15:0 and iso-C 16:0 . Phylogenetic analyses based on 16S rRNA gene sequences showed that strain GA224 T fell within the genus Yonghaparkia, the highest 16S rRNA gene sequence similarity values (98.60%) being obtained with respect to Yonghaparkia alkaliphile KSL-113 T . The draft genome of strain GA224 T comprised 2,495,189 bp with a G+C content of 72.17 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain GA224 T and phylogenetically related Yonghaparkia species were lower than 95% and 70%, respectively. Based on the phenotypic, chemotaxonomic and genomic data, strain GA224 T represents a novel species, for which the name Yonghaparkia aerolata sp. nov. is proposed, with GA224 T as the type strain (= GDMCC 1.2165 T = JCM 34462 T ).


Introduction
The genus Yonghaparkia, a member of the family Microbacteriaceae, was rst proposed by Yoon (2006). The strain Yonghaparkia alkaliphile was isolated from an alkaline soil in Kwangchun, Korea. At the time of writing, the genus Yonghaparkia comprised only one species with validly published name (https://lpsn.dsmz.de/genus/yonghaparkia). The genus Yonghaparkia is characterized as strictly aerobic Gram-positive, non-spore-forming short rods or rods bacteria. Members of genus Yonghaparkia contain MK-12 and MK-11 as the dominant menaquinone, and B2γ as the major peptidoglycan type in cell-wall. The fatty acid pro le consists of branched and straight-chain fatty acids, with anteiso-C 15:0 and iso-C 16:0 being the most dominant. The DNA G+C content is 71.1−71.6 mol%. Yonghaparkia-related sequences have been detected mainly in shale or soil environments (Kou et al. 2018;Li et al. 2014;Włodarczyk et al. 2018). The isolation and characterization of members of the genus, therefore, would be helpful for a better understanding of their ecology and taxonomy due to the scarcity of cultured bacterial isolates (Overmann et al. 2017). In this study, a presumably novel bacterial strain belonging to the genus Yonghaparkia, designated strain GA224 T , was isolated from an electronic waste associated bioaerosols in Guiyu, China and it was characterized taxonomically using a polyphasic approach.

Sampling and isolation
An electronic waste associated bioaerosol sample collected from Guiyu, Guangdong province of China (23.32N, 116.36E) in November 2019, was used to isolate bacterial strains (Pan et al. 2021).
Bioaerosols were collected by six-stage Andersen Cascade Impactors with 10 × diluted nutrient agar (NA) followed by aerobic incubation at 37 ℃ for 48 h. Single colonies were separated and puri ed by repeated streaking on fresh 2 × diluted NA plates. Only one light yellow isolate GA224 T , a unique alkalophilic microorganism compared to other potentially novel bacteria, was selected for further study (Kim et al. 2014). The isolate was routinely cultured on 2 × diluted NA at 37 °C and preserved as a suspension in 2 × diluted nutrient broth (NB) with glycerol (15%, w/v) at − 80 °C. Strain GA224 T has been deposited to the Japan Collection of Microorganisms (JCM) and Guangdong Microbial Culture Collection Center (GDMCC). For the comparative study, the reference strain Yonghaparkia alkaliphile KCTC 19126 T was obtained from GDMCC and tested using the same laboratory conditions as for strain GA224 T .

Morphological, physiological and biochemical characteristics
The morphological, physiological and biochemical characteristics of strain GA224 T were characterized after 48 h of incubation at 37 ℃ on 2 × diluted NA or 2 × diluted NB with the pH adjusted to 8.0. Cell morphology, size, shape, agellation and Gram staining were examined by transmission electron microscope (Harris 2015) and light microscopy, using freshly grown cells. Growth at various temperatures (4,15,25,30,37, and 40 °C) and numerous pH conditions (pH 4.5−10.5, 0.5 pH unit interval) were evaluated in 2 × diluted NB after 48 h of incubation at 37 °C. Two different buffers, 1M HCl (for pH 4.5-6.5), and 1M NaOH (for pH 7.0-10.5) were used for pH test. Salinity tolerance was evaluated in 2 × diluted trypticase soy broth supplemented with 0, 0.5 and 1.0−5.0% (w/v), at intervals of 1.0% after 48 h of incubation at 37 °C. Growth was determined by checking the optical density at 600 nm. Growth under anaerobic conditions was determined after incubation in an anaerobic incubator on 2 × diluted NA (pH 8.0). Catalase, and oxidase activities were determined using 3% (v/v) H 2 O 2 and 1% (v/v) dimethyl-pphenylenediamine hydrochloride reagent, respectively (Harrigan and McCance 1966). Enzyme activities and utilization of carbon sources were further determined using the commercial kits API ZYM (Biomerieux) and GEN III MicroPlate system (BIOLOG), according to the manufacturer's instructions. Antibiotic sensitivity was tested by broth microdilution method (Balouiri et al. 2016) with 2 × diluted NB containing the following antibiotics: streptomycin sulphate, gentamycin solution, kanamycin sulfate, vancomycin hydrochloride, tetracycline hydrochloride, cipro oxacin hydrochloride, cephalosporin, penicillin G, imipenem, and ampicillin.

Chemotaxonomy analysis
For cellular fatty acid analysis, 40 mg of cells were harvested from 2 × diluted NA plates after incubation for 48 h at 37 ºC. Fatty acids were extracted, methylated, and saponi ed according to the manufacturer's protocol of Microbial Identi cation System and identi ed by gas chromatography (Sasser 1990;Sun et al. 2018). The peaks obtained were then labeled, and the equivalent chain length values were computed by the Sherlock software. Menaquinones were extracted as described by Collins (Collins et al. 1977) and analyzed by reverse phase high-performance liquid chromatography (Ranmadugala et al. 2018). The polar lipids pro le was analyzed by extracting cells with methanol-chloroform-saline (2:1:0.8, v/v/v) from 1 g of freeze-dried bacterial cells. Separation of lipids was performed by two-dimensional chromatography on a silica gel thin-layer chromatography plate using chloroform-methanol-water (65:25:4, v/v/v) in the rst dimension and chloroform-methanol-acetic acid-water (80:12:18:5, v/v/v/v) in the second dimension (Park et al. 2018). The total polar lipids pro le was detected using several color developing agents. The dodecamolybdophosphoric acid was used for detecting total lipid, molybdenum blue for phosphate, ninhydrin for free amino group, Dragendorff reagent for quaternary nitrogen and methyl naphthol for glycolipids as described by Tindall (Tindall 1990  The multiple alignments were accomplished using the CLUSTAL W program. The phylogenetic trees were constructed using both the neighbor-joining method (Saitou and Nei 1987) and the maximum-likelihood method (Felsenstein 1981)  The draft genome sequence of strain GA224 T was performed with Illumina HiSeq sequencing system with paired-end DNA libraries that were prepared at Magigene Company (Shenzhen, China). The reads of each data set were ltered, and de novo assemblies were performed by using the software SPAdes version 3.5.0 (Bankevich et al. 2012). The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Hyatt et al. 2010). The sequence is available from the NCBI under the accession number JABANT000000000. To estimate the degree of pairwise relatedness between GA224 T , and close relatives, average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) was calculated.

Chemotaxonomic Properties
Cellular fatty acid of strains GA224 T and Y. alkaliphile KCTC 19126 T were shown in Table 2. The major fatty acids components (> 10% of total fatty acids) of strain GA224 T were identi ed as anteiso-C 15:0 (35.07%) and iso-C 16:0 (20.53%), followed by anteiso-C 17:0 (9.33%) and C 16:0 (8.37%). A similar fatty acid pro le was found in the reference strain Y. alkaliphile KCTC 19126 T , but there were quantitative differences when cultivated under the same conditions. The predominant menaquinone was determined as MK-12 in strain GA224 T , which is a common characteristic in the genus Yonghaparkia. However, Strain GA224 T differed from Y. alkaliphile KCTC 19126 T in minor menaquinone, with MK-11 (22.49%) and MK-7 (18.98%) containing similar proportions. The polar lipids presented in these two strains were diphosphatidylglycerol, phosphatidylglycerol, unidenti ed phospholipids and unidenti ed glycolipids, while unidenti ed lipid was also detected in strain GA224 T (Fig. 1d-f). Analysis of peptidoglycan showed that strain GA224 T , as well as the reference strain, contained glutamic acid, glycine and alanine in different ratios 1.0:0.73:0.41. The 2,4-diaminobutyric acid was presented in both of their cell-wall (Fig. 1g). In the neighbor-joining tree based on 16S rRNA gene sequences, strain GA224 T clustered within the phylogenetic lineage of the genus Yonghaparkia at a bootstrap resampling value of 97% (Fig. 2). The relationship between strain GA224 T and Yonghaparkia alkaliphila KSL-113 T was maintained in trees generated with the maximum-likelihood and maximum-parsimony algorithms. Based on the results of the phylogenetic analysis and sequence similarity threshold of the 16S rRNA (98.65%) (Kim et al. 2014), strain GA224 T may represent a novel species of the genus Yonghaparkia.
Comparison of the draft genome of strain GA224 T with those of its closest phylogenetic neighbors  (Table 3), which were much lower than the cutoff point of 70% for the delineation of a novel species (Goris et al. 2007).

Conclusion
In conclusion, based on the phenotypic analysis including morphology, biochemistry and chemotaxonomy and genotypic properties, the strain GA224 T is classi ed as a novel species within the genus Yonghaparkia, for which the name Yonghaparkia aerolata sp. nov. has been proposed.
The cells stain Gram-positive and are facultatively anaerobic, short rod-shaped, 0.2-0.4 µm wide and 0.5-1.1 µm long. Colonies on 2 × diluted NA (pH 8.0) are light yellow, opaque, circular and smooth, with regular edges, no halo ring and approximately 1.6-1.8 mm in diameter after incubation for 48 h at 37 ℃. Growth occurs at 25-40 ℃ with an optimal of 37 ℃ and from pH 6.5-8.5 (optimum pH 8). Growth occurs in the presence of 0-5.0% (w/v) NaCl at 37 ℃ with the optimal NaCl concentration for growth 1.0% (w/v), and it is both catalase and oxidase positive. The major fatty acids (> 10% of total fatty acids) are anteiso-C 15:0 and iso-C 16:0 . The major polar lipids are diphosphatidylglycerol, phosphatidylglycerol, unidenti ed phospholipids, unidenti ed glycolipids and unidenti ed lipid. The peptidoglycan contains glutamic acid, glycine, alanine and 2,4-diaminobutyric acid. The DNA G+C content of the type strain is 72.17 mol%.
The type strain, GA224 T (= GDMCC 1.2165 T = JCM 34462 T ), was isolated from an electronic waste associated bioaerosols from Guangdong province of China.